Development of an Indirect Enzyme-Linked Immunosorbent Assay for Seromonitoring Contagious Bovine Pleuropneumonia Using Recombinant Lipoprotein LppQ of Mycoplasma mycoides subsp mycoides SC as Antigen

Mycoplasma mycoides subsp mycoides SC （MmmSC） is the etiological agent of contagious bovine pleuropneumonia （CBPP）. The lipoprotein LppQ encoded by lppQ gene is specific to MmmSC and is found in the type strain and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains. No serological cross-reactions were observed with the related mycoplasmas of the Mycoplasma mycoides cluster. The N-terminal domain of the mature lipoprotein LppQ is hydrophilic, and it induces a strong, specific, early, and persistent immune response in naturally and experimentally infected animals. Mycoplasma-specific TGA （Trp） codons are utilized as stop codons in most other organisms. The lppQ N-terminal fragment from MmmSC HVRI X strain, the Chinese strain for CF antigen production, was mutated with one-step overlapping extension PCR. Sequence analysis confirmed the successful mutation from A to G in codon 198 in the lppQ gene. The fragment containing the mutation site was subcloned into the pET32a expression vector. The recombinant protein with molecular weight of 42 kDa was purified using the Ni-NTA His.Bind purification kit, with a purity of up to 95%. Western blot indicated that the standard positive serum of CBPP could react with the recombinant protein. The purified protein was diluted to 0.35 μg mL^-1, and coated to microtiter enzyme-linked immunosorbent assay （ELISA） plates. Indirect ELISA reaction conditions were optimized. The value of P/N was determined to be 4.8 （0.934/0.193）, the sensitivity to be 95.8% （46/48）, and the specificity to be 98.9% （161/163）. 3 817 cattle serum samples from three different provinces were detected by the indirect ELISA and CFT. The Kappa value is 0.63, which is middle or high agreement between the two methods.

Microcystin-LR detection based on indirect competitive enzyme-linked immunosorbent assay

Microcystins(MCs)are a group of closely related toxic cyclic heptapeptides produced by common cyanobacte-ria,which cause lots of accidents and threatens human health.In this paper,an indirect competitive enzyme-linked immu-nosorbent assay(ic-ELISA)was established and used to detect microcystin-LR(MC-LR)in drinking and surface waters.The concentration of coating antigen was 5 mg/mL,the dilution of monoclonal antibody MC10E7 was 1:3000,the dilution of enzyme tracer(goat anti-mouse IgG-peroxidase)was 1:3000,the standard concentration of MC-LR ranged from 0.001 mg/L to 30 mg/L,and o-phenylenediamine was used as substrate.The assay showed high relativity with high performance liquid chromatography(HPLC)with a correlation coefficient of more than 99%.The relative standard deviation was less than 10%,the detection limit was achieved down to 0.01 mg/L and up to 5.1 mg/L.The quantitative detection range was from 0.03 mg/L to 3 mg/L,and the antibody had high specificity for[4-arginine]microcystins.It performed well in spite of the influence of the real samples.

A sensitive and convenient enzyme-linked immunosorbent assay method in serum MG7 antigen detection in gastric cancer

Objective To explore a highly sensitive and highly specific method to detect the serum MG7 antigen(Ag)level for early gastric cancer diagnosis.Methods The serum MG7-Ag level was detected by enzyme-linked immunosorbent assay(ELISA)method in 116 preoperative gastric cancer patients,63 postoperative gastric cancer patients,41 patients with precancerous lesion,37 pa-

Development of ELISA and immunochromatographic assay for ofloxacin

Two rapid, sensitive and reliable immunoassay methods, namely competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) and colloidal gold-based immunochromatographic assay (CGIA), were developed to detect ofloxacin (OFL). The linear range of the CI-ELISA was from 0.5 to 128 ng/mL with a limit of detection (LOD) of 0.35 ng/mL. Good recoveries were obtained in analyzing simulated swine urine samples. The CGIA could accurately estimate OFL at concentrations as low as 10 ng/mL in less than 10 min, and test results were read visually without any instrument.

间接竞争酶联免疫分析法快速检测决明子中黄曲霉毒素B_(1)的研究

目的:建立适用于中药决明子中黄曲霉毒素B_(1)(aflatoxin B_(1),AFB_(1))快速检测的间接竞争酶联免疫分析法(indirect competitive enzyme linked immunosorbent assay,ic-ELISA)。方法:首先采用棋盘滴定法优化抗原抗体最佳浓度,建立抑制曲线,然后以显色值和灵敏度为评价指标筛选样品的提取和稀释方法。对所建立的方法进行方法学考察,最后用于实际样品检测。结果:经过优化,确定样品的最佳提取溶剂为乙腈-甲醇溶液(90∶10,v/v),稀释溶液为甲醇-PBS溶液(10∶90,v/v),稀释倍数为20倍。方法的半数抑制浓度为0.078 ng/mL,线性范围为0.016~0.25 ng/mL,回收率为85.00%~109.2%,RSD为0.45%~9.08%。采用所建立的方法对25批决明子进行了检测,经液相色谱-串联质谱法确证,超过限量标准的样品的假阳性率小于5%。结论:本文所建立的ic-ELISA灵敏、简便,适用于决明子中AFB_(1)的快速筛查,但仍存在一定的假阳性率,后续还需要对产生干扰的基质因素进行研究并消除,进一步提高检测的准确度。

Immunodiagnostic efficacy of detection of Schistosoma japonicum human infections in China:a meta analysis

Objective:To assess the diagnostic efficacy of the currently most widely used indirect hemagglutination assay(IHA) and enzyme-linked immunosorbent assay(ELISA) for detection of Schistosoma japonicum human infections.Methods:A comprehensive search was undertaken from China National Knowledge Infrastructure,Wanfang Database.VIP Database,PubMed. Cochrane Library,Science Citation Index Expanded.Proquest,and the inclusion and exclusion criteria were strictly settled.The funnel plot was used to assess the publication bias.Cochran’s Q test was employed to measure the homogeneity between studies,a summary receiver operating characteristic(SROC) curve was used to compare the diagnostic accuracy between the IHA and ELISA qualitatively by means of the Weighted Least Square method,the Ordinary Least Square method and the Robust regression method,and the diagnostic odds ratio(DOR) was drawn to compare the accuracy quantitatively.Results:Out of 785 publications,19 papers were eventually selected for analysis.Literature finality assessment indicated that minor publication bias existed in studies pertaining IHA test,but no bias was found in literatures regarding ELISA test.The heterogeneity test showed a heterogeneity between studies was present(χ~2 =466.07 and 34.67. both P values【0.0001).The areas under the SROC curves of IHA were all higher than that of ELISA test using the three methods(Weighted Least Square method:0.766 vs.0.695.Ordinary Least Square method:0.826 vs.0.741.Robust regression:0.815 vs.0.715).The TPR* values for IHA and EUSA were 0.710.0.759.0.749.and 0.650.0.686 and 0.666.respectively,and OR values were 5.997.9.937.8.893.and 3.432.4.784 and 3.959.respectively.The DOR of IHA was 9.41(95% CI:4.88-18.18).and 4.78(95%CI:3.21-7.13) for ELISA.Conclusions:All above results revealed that the diagnostic performance of IHA is better than that of ELISA.However,taking into account their unsatisfactory diagnostic value in areas with low infection intensity,a search for a better diagnostic test that can be applied in field situations in China should be given high priority.

间接竞争ELISA法检测黄连解毒汤中黄芩苷在大鼠脑脊液中的分布

目的检测黄连解毒汤灌胃给药后黄芩苷在大鼠脑脊液中的分布。方法通过考察线性关系、精密度、回收率等方法学考察指标,建立基于黄芩苷单克隆抗体的间接竞争ELISA(icELISA)法;通过建立的icELISA法,检测大鼠灌胃给药后1小时、2小时、3小时和4小时脑脊液中黄芩苷的含量。结果在大鼠脑脊液中建立的黄芩苷标准曲线为y=0.1069ln(x)+1.0144(R 2=0.9885),浓度范围为3.2~2000 ng/mL,在400、80、3.2 ng/mL三个浓度的精密度分别为13.14%、6.65%和6.04%(RSD≤15%),回收率分别为91.35%、87.04%和117.67%(SD≤15%)。给药后1小时、2小时、3小时和4小时脑脊液中黄芩苷的平均含量分别为(60.53±18.05)、(223.32±29.72)、(121.35±31.93)、(4.46±2.54)ng/mL。结论在脑脊液中建立的黄芩苷icELISA检测法灵敏度高、精密度和回收率能够满足生物样品的检测要求,可以作为黄芩苷的检测方法。在脑脊液中可以检出黄芩苷,给药后2小时黄芩苷在脑脊液中的浓度最高,然后随时间逐渐降低,说明黄芩苷可以透过血脑屏障从而分布到脑脊液中,为黄芩苷治疗脑部疾病提供了直接的实验依据。

基于抗大豆苷单克隆抗体的Ic-ELISA方法的建立

目的：建立大豆苷的快速免疫分析检测方法。方法：该研究通过大豆苷单克隆抗体与葛根素、芦丁等共9种中药小分子的交叉反应证明该抗体具有特异性,并以制备出的大豆苷特异性单克隆抗体为基础,建立了大豆苷间接竞争酶联免疫分析方法（Ic-ELISA）,并用此方法检测大豆中大豆苷的含量。结果：抗体与葛根素的交叉反应率为115%,与芦丁、甘草酸、栀子苷等小分子无交叉反应。该方法线性范围为10~10 000 ng·m L^-1,孔内差和板间差均小于6.3%,平均回收率为105.4%%。采用该方法检测大豆中大豆苷的含量,所得结果与HPLC法的结果一致。结论：该研究建立了大豆苷的Ic-ELISA方法,可为含大豆苷的中药及复方的质量控制分析提供更加快速灵敏的检测方法。

Effect of Puerarin on the Pharmacokinetics of Baicalin in Gegen Qinlian Decoction (葛根芩连汤) in Mice

Objective： To study the pharmacokinetics of puerarin（PUE） in Gegen Qinlian Decoction（葛根芩连汤, GQD）, and the effects of PUE dosage variations on the pharmacokinetics of baicalin（BAL） in mice. Methods： GQD is composed of the concentrated granules of four Chinese herbs. Three dosages with different levels of PUE, including GQD, GQD co-administered with PUE, and GQD co-administration with two times the amount of PUE, were used to research the pharmacokinetics of PUE and BAL in mice. The indirect competitive enzyme-linked immunosorbent assay（ic ELISA） methods based on an anti PUE-monoclonal antibody（MAb） and BAL-MAb were employed to determine the concentration of PUE and BAL in mice blood. Results： After the co-administration of GQD with PUE, the area under the curves（AUC0-14 h） of PUE increased 2.8 times compared with GQD. At the dose of GQD co-administration at two times that of PUE, the AUC0-14 h of PUE was almost equal to that of GQD co-administration of PUE, showing non-linear pharmacokinetics. The AUC0-48 h of BAL showed a good dose-related increase of PUE（r=0.993） in the range from 100 to 300 mg/kg, indicating that PUE dramatically affects the absorption of BAL in mice. There was no significant difference in the other pharmacokinetic parameters, such as the first time of maximum concentration（Tmax）, the second Tmax, or the mean residence time. Conclusions： The ic ELISA methods were successfully applied to pharmacokinetic studies of PUE and BAL in GQD in mice. The dosage variability of PUE of the main ingredient in GQD affects its own pharmacokinetic characteristics and the absorption characteristics of BAL.